Crucial to our understanding of synaptic function is the concept of the probability release (p), which corresponds to the probability that a presynaptic action potential results in vesicle fusion at a single release site. Much is known about the mechanisms that change p from its initial value, but much less is known about the mechanisms that set and maintain the initial p of a synapse. Factors such as the density and distribution of presynaptic calcium channels, calcium sensitivity of the release machinery, and various presynaptic signaling cascades are thought to influence initial p. However, it is not understood which of these factors are most important at specific synapses and how these factors may interact to set initial p. This proposal will address one question regarding the regulation of initial p: is tonic presynaptic inhibition important for setting initial p? This project will test the hypothesis that tonic inhibition by multiple presynaptic receptors produces a large reduction in initial p. Preliminary data suggests that multiple presynaptic G-protein coupled receptors (GPCRs) at the cerebellar parallel fiber-Purkinje cell (PF-PC) synapse work together to produce a greater than two-fold reduction in initial p. This contrasts with the very small or negligible effects of individual presynaptic GPCRs on initial p in isolation. This application proposes experiments to characterize GPCR-mediated tonic presynaptic inhibition and to test for its potential physiological significance. The primary techniques used will be (1) whole-cell voltage clamp recordings, (2) bulk presynaptic calcium imaging in slices, (3) local field potential recordings in vivo, and (4) bulk presynaptic calcium imaging in vivo. Based on preliminary experiments, this proposal will focus on the effects of presynaptic adenosine (A1) receptors, endcannabinoid (CB1) receptors, GABAB receptors, and metabotropic glutamate type 4 (mGlu4) receptors. The first and second specific aims will characterize the extent of tonic GPCR-mediated inhibition and search for presynaptic mechanisms by which this inhibition may occur. The third aim will test for potential physiological relevance of tonic presynaptic inhibition. This project is expected to provide considerable insight into the regulation of release probability by endogenous signaling molecules and has the potential to implicate activity-dependent mechanisms by which this regulation may occur. Such insights are also likely to provide insight into mechanisms regulating motor learning.